C2C12 myoblasts were grown to confluence in DMEM supplemented with 10% FCS. They were induced to differentiate into myotubes, by switching to DMEM containing 2% horse serum. Primary cultures of human skeletal muscle cells were initiated from satellite cells of quadriceps samples obtained from organ donors (3 men, 27 ± 7 years, 23 ± 1.7 kg/m2). Differentiated myotubes were prepared according to the procedure previously described in details (37). Myotubes were then cultured for 96 hours, with or without H2O2 (100µM), with mannitol (25mM) or glucose (25mM), and with BSA (1%) or palmitic acid (200µM), in the presence or not of N-acetylcysteine (NAC, 10mM).
ROS production in C2C12 cells
ROS production was detected by the nitroblue tetrazolium (NBT, Sigma) assay. NBT is reduced by ROS to a dark-blue, insoluble form of NBT called formazan. After treatment, myotubes were incubated for 90 minutes in DMEM containing 0.2% NBT. Formazan was dissolved in 50% acetic acid, and the absorbance was determined at 560 nm. Optical density values were normalized by protein levels.
Statistical analysis
The student’s t-test was used to analyze the difference between control and experimental groups. P< 0.05 was considered to be significant.
Acknowledgements
This work was supported by grants of INSERM, the French National Research Agency (ANR-05-PCOD-012) and the French National Program on Diabetes Research (to J.R). C.B. received fellowships from Rhônes-Alpes region. The authors thank the imagery centre of Laennec Faculty and the IFR62, for access to platforms.
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Figure Legends
Figure 1: Decreased mitochondrial density in skeletal muscle of 16 week HFHSD mice. A: mtDNA quantity calculated as the ratio of COX1 to cyclophilin A DNA levels, determined by real time PCR, in skeletal muscle of SD and HFHSD mice, after 4 and 16 weeks of diet (n=6). Note that the y-axis scale is between 0.5 and 0.8. B: mRNA expression of mitochondria-encoded COX1 and COX3 genes, determined by real-time RT-PCR in skeletal muscle of 16 week HFHSD mice (n=6). Results were normalized by the mean value of the 16 week SD condition set to one unit. C: mitochondrial density assessed by electron microscopy, in skeletal muscle from SD and HFHSD mice, after 4 and 16 weeks of diet. D: Quantification of subsarcolemmal and intermyofibrillar mitochondria number per image area in gastrocnemius muscle of 16 week HFHSD mice (analysis of 5 images in 3 mice per group). Results were normalized by the mean value of the 16 week SD condition set to one unit. E- Citrate synthase (CS) activity in isolated mitochondria from gastrocnemius of SD and HFHSD mice, after 4 and 16 weeks of diet (n=6). * p<0.05, ** p<0.01 relative to SD mice, $ p<0.05 relative to 4 week SD mice.
Figure 2: Expression of genes implicated in mitochondrial biogenesis and in mtDNA replication. mRNA levels of mitochondrial biogenesis (A) and mtDNA replication (B) genes, determined by real-time RT-PCR, in gastrocnemius muscle of SD and HFHSD mice, after 4 and 16 weeks of diet (n=6). Results were expressed as fold change versus the SD conditions set to one unit (dotted line). * p< 0.05. PGC1: PPAR gamma coactivator 1, NRF: nuclear respiratory factor, mtTFA: mitochondrial transcription factor, ERRestrogen-related receptor , Mfn2: mitofusin 2, POLG: polymerase gamme, SSBP1: single strand binding protein.
Figure 3: Alterations in mitochondrial structure in skeletal muscle of 16 week HFHSD-fed mice. A-B: Transmission electronic microscopy images (magnification x 25,000 (A) and x 100,000 (B)) of subsarcolemmal and intermyofibrillar mitochondria in gastrocnemius muscle of 16 weeks SD and HFHSD-fed mice. C: Quantification of subsarcolemmal and intermyofibrillar mitochondria area in gastrocnemius muscle of 16 week HFHSD mice (analysis of 5 images in 3 mice per group). Results were normalized by the mean value of the 16 week SD condition set to one unit.* p<0.05, **p<0.01.
Figure 4: Chronic HFHSD feeding induces an oxidative stress in skeletal muscle. A: Immunoblots showing total protein carbonylation in gastrocnemius muscle of SD and HFHSD mice, after 4 and 16 weeks of diet. B: mRNA levels of oxidant stress-related genes, determined by real-time RT-PCR, in gastrocnemius muscle of SD and HFHSD mice, after 4 and 16 weeks of diet (n=6). Results were expressed as fold change versus the SD conditions set to one unit (dotted line). * p< 0.05. C: Immunoblot showing cytochrome C protein in mitochondrial (MF) and cytosolic (CF) fractions of gastrocnemius muscle of SD and HFHSD mice, after 4 and 16 weeks of diet. D: Caspase 3 activity measured in gastrocnemius muscle of 4 and 16 week HFHSD mice (n=6). Results were normalized by the mean values of the 4 and the 16 week SD conditions, respectively. * p<0.05. UCP: uncoupling protein, GSR: glutathione reductase, GPx: glutathione peroxidase, CAT: catalase, SOD: superoxide dismutase, Prdx: peroxiredoxin.
Figure 5: Streptozotocin-induced oxidative stress alters mitochondria density and structure in skeletal muscle. A: Immunoblots showing total protein carbonylation in gastrocnemius muscle of control (Co), streptozotocin-treated (STZ) and insulin-treated STZ mice (STZ+INS). B: Immunoblot showing cytochrome C protein in mitochondrial (MF) and cytosolic (CF) fractions of gastrocnemius muscle of Co, STZ and STZ+INS mice. C: mtDNA copy number was calculated as the ratio of COX1 to cyclophilin A DNA levels, determined by real time PCR, in skeletal muscle of Co, STZ, STZ+INS, and phlorizin-treated STZ mice (STZ+PHL) (n=6). Note that the y-axis scale is between 0.5 and 1. Results were normalized by the mean value of the control condition set to one unit. * p< 0.01 STZ vs. Co, # p<0.05 compared to STZ mice. D: Transmission electronic microscopy images (x 25,000) of gastrocnemius muscle from Co, STZ, STZ+INS, and STZ+PHL mice, pointing both subsarcolemmal and intermyofibrillar mitochondria.
Figure 6: Antioxidant treatment restores mitochondrial alterations in STZ mice. A: Plasma H2O2 levels in control (Co) and streptozotocin mice, treated (STZ+NAC) or not (STZ) with N-acetylcysteine (10mM in drinking water). B: Immunoblots showing total protein carbonylation in gastrocnemius muscle of Co, STZ and STZ+NAC mice. C: mtDNA copy number was calculated as the ratio of COX1 to cyclophilin A DNA levels, determined by real time PCR, in skeletal muscle of Co, STZ and STZ+NAC mice. Note that the y-axis scale is between 0.5 and 1. Results were normalized by the mean value of the control condition set to one unit. * p< 0.01 STZ vs. Co, # p<0.05 compared to STZ mice. D: Transmission electronic microscopy images (x 25,000) of gastrocnemius muscle of STZ and STZ+NAC mice, pointing both subsarcolemmal and intermyofibrillar mitochondria.
Figure 7: ROS-induced mitochondrial alterations in C2C12 muscle cells. A- ROS production, measured by NBT reduction, measured in differentiated C2C12 myotubes incubated with glucose (25mM) or palmitate (200µM), in presence or not of NAC (10mM), for 96 hours (n=4). Data are expressed relative to respective control (dotted line). B- Effect of H2O2 (0.1mM) , glucose (25mM) and palmitate (200µM) on mtDNA levels in differentiated C2C12 myotubes. Myotubes were treated for 96 hours in presence or not of 10mM NAC (n=4). Data are expressed relative to control condition (dotted line). C: Citrate synthase (CS) activity measured in total lysates of myotubes treated for 96 hours with H2O2 (0.1mM), glucose (25mM) or palmitate (200µM), in presence or not of NAC (10mM) (n=4). D: mRNA levels of POLG2, SSBP1 and PGC-1 genes, determined by real-time RT-PCR, in H2O2, glucose or palmitate-treated myotubes, in presence or not of NAC, for 96 hours (n=4). All results are expressed as fold change relative to the values of untreated cells set to one unit (dotted line). *p<0.05, ** p<0.01 vs. respective control. # p<0.05 with and without NAC for each treatment.
Table 1
Characteristics of the mice
| SD/HFHSD protocol | STZ/INS protocol |
|
SD 4w
|
HFHSD 4w
|
SD16w
|
HFHSD16w
|
Co
|
STZ
|
STZ+INS
| |
Body weight (g)
|
24.08 ± 0.38
|
28.9 ± 0.6**
|
27.7 ± 0.36
|
44 ± 1.02**
|
23.9 ± 0.1
|
19.6 ± 0.1§
|
22 ± 0.4$
|
Fat weight (g)
|
0.25 ± 0.02
|
1.1 ± 0.07**
|
0.61 ± 0.05
|
2.5 ± 0.08**
|
nd
|
nd
|
nd
|
Glucose (mg/dl)
|
167 ± 4.7
|
170 ± 4.7
|
149 ± 4.1
|
210.7 ± 7**
|
160 ± 4
|
598.5 ± 21.6§§
|
484.3 ± 22$$
|
Insulin (ng/ml)
|
0.87 ± 0.2
|
1.5 ± 0.4*
|
0.8 ± 0.22
|
7.4 ± 1.6**
|
0.8 ± 0.2
|
0.3 ± 0.02§§
|
15.4 ± 3.3$$
|
Leptin (pg/ml)
|
3.1 ± 0.3
|
5.5 ± 1.7*
|
8.4 ± 0.9
|
55 ± 3.7**
|
nd
|
nd
|
nd
|
TG (g/l)
|
0.3 ± 0.07
|
0.2 ± 0.03
|
0.16 ± 0.03
|
0.4 ± 0.06**
|
nd
|
nd
|
nd
|
FFA (mM)
|
0.5 ± 0.04
|
0.6 ± 0.13
|
0.45 ± 0.06
|
0.9 ± 0.1*
|
0.11 ± 0.01
|
0.12 ± 0.05
|
ud
|
H2O2 (µM)
|
29.9 ± 8
|
33.02 ± 5.7
|
28.8 ± 3.4
|
44 ± 4.5*
|
nd
|
nd
|
nd
|
Data represent the means ± sem of 10-20 mice per group.
* p<0.05, ** p<0.001 vs. the respective SD mice.
§ p<0.05, §§ p< 0.001 between Co and STZ mice.
$ p<0.05, $$ p<0.001 between STZ and STZ+INS mice.
nd: not determined, ud: undetected, SD: standard diet, HFHSD: high fat and high sucrose diet, Co: control, STZ: streptozotocin-treated mice, STZ+INS: insulin-treated STZ mice, TG: triglyceride, FFA: free fatty acids.
Table 2
Respiration rates and respiration control ratio (RCR) in permeabilized muscle fibers from SD and HFHSD mice, after 4 and 16 weeks of feeding.
|
Respiration rates (nat O/(min.mg dry weight)
|
|
|
SD 4w
|
HFHSD 4w
|
SD 16w
|
HFHSD 16w
|
Glutamate /Malate
|
State 4
|
2.45 ± 0.3
|
2.55 ± 0.2
|
2.23 ± 0.1
|
1.62 ± 0.2 *
|
|
State 3
|
6.43 ± 0.9
|
6.15 ± 0.8
|
6.34 ± 0.4
|
4.5 ± 0.4 *
|
|
RCR
|
2.79 ± 0.5
|
2.47 ± 0.3
|
2.86 ± 0.2
|
2.87 ± 0.3
|
Succinate/Rotenone
|
State 4
|
5.02 ± 0.6
|
6.61 ± 0.9
|
5.25 ± 0.5
|
5.55 ± 0.7
|
|
State 3
|
10.7 ± 1.4
|
14.4 ± 1.2
|
11.85 ± 1.4
|
11.1 ± 0.9
|
|
RCR
|
2.79 ± 0.5
|
2.47 ± 0.4
|
2.86 ± 0.2
|
2.87 ± 0.3
|
Octanoyl-carnitine/Malate
|
State 4
|
1.86 ± 0.1
|
1.36 ± 0.1
|
1.4 ± 0.1
|
0.99 ± 0.1
|
|
State 3
|
3.93 ± 0.4
|
3.52 ± 0.5
|
3.38 ± 0.5
|
1.73 ± 0.2 *
|
|
RCR
|
2.11 ± 0.18
|
2.66 ± 0.5
|
2.29 ± 0.4
|
1.85 ± 0.2
|
Palmitoyl-carnitine/Malate
|
State 4
|
1.18 ± 0.2
|
1.49 ± 0.1
|
1.47 ± 0.2
|
1.01 ± 0.1
|
|
State 3
|
2.19 ± 0.3
|
2.99 ± 0.3
|
3.68 ± 0.6
|
1.72 ± 0.26 *
|
|
RCR
|
1.95 ± 0.2
|
2.07 ± 0.3
|
2.65 ± 0.4
|
2.08 ± 0.3
|
Respiration rates were measured on saponin-skinned fibers as described under Materials and methods. State 4 was the control state of respiration in presence of actractyloside and state 3 was the ADP-stimulated respiration. RCR was the respiratory control ratio (state3/state 4). Values are means ± sem for 6 animals per group. * p<0.01 different from the respective SD mice.
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